The molecular complexity of hepatic microsomal cytochromes P450 (P450) in the rat is not only a consequence of multiple isozymes but also involves electrophoretically distinct allozymes and post-translationally truncated forms of these hemoproteins. Furthermore, regulatory variants for high and low level of several isozymes have been demonstrated and alleles for specific allozymic and regulatory mutants have been shown to characterize particular strains of inbred animals. In addition to genetic polymorphisms already reported for P-450s b, e, g and h, we have recently discovered apparent non-allelic, allelic and regulatory polymorphisms for other members of the P450II family (i.e., P450s k, a and RIM2, respectively) and these will be further verified in the initial phase of the proposed research. A major goal of this proposal is to comprehensively elucidate genetic variation of P450 function. Therefore, we plan to test whether structural polymorphisms of four P450 isozymes are associated with catalytic differences. At least two electrophoretic variants each for P450s b, e, h, and k as well as post-translationally modified forms of P450h will be purified from previously characterized strains of inbred rats and assayed for catalytic activity in reconstituted systems. Aflatoxin B1 (AFB1), steroids and an increasingly complex panel of substrates will be tested. In addition to AFB1, other substrates of biomedical interest (e.g. valproic acid, vitamin D3, bile acid precursors, etc.) are proposed for studies that should elucidate pharmacologically/physiologically relevant catalytic differences between P450 isozymes and their polymorphic forms. If catalytic differences are observed between P450 allozymes then their sequences will be determined from cDNAs in an effort to establish structure/function relationships. The other major goal of the proposed research is to employ already characterized inbred strains of rat in genetic crosses aimed at the chromosomal location vis a vis four other P450II genes already mapped should provide new information regarding the evolution of this complex gene family.